Nebulin binding impedes mutant desmin filament assembly.

Abstract

Desmin intermediate filaments (DIFs) form an intricate meshwork that organizes myofibers within striated muscle cells. The mechanisms that regulate the association of desmin to sarcomeres and their role in desminopathy are incompletely understood. Here we compare the effect nebulin binding has on the assembly kinetics of desmin and three desminopathy-causing mutant desmin variants carrying mutations in the head, rod, or tail domains of desmin (S46F, E245D, and T453I). These mutants were chosen because the mutated residues are located within the nebulin-binding regions of desmin. We discovered that, although nebulin M160-164 bound to both desmin tetrameric complexes and mature filaments, all three mutants exhibited significantly delayed filament assembly kinetics when bound to nebulin. Correspondingly, all three mutants displayed enhanced binding affinities and capacities for nebulin relative to wild-type desmin. Electron micrographs showed that nebulin associates with elongated normal and mutant DIFs assembled in vitro. Moreover, we measured significantly delayed dynamics for the mutant desmin E245D relative to wild-type desmin in fluorescence recovery after photobleaching in live-cell imaging experiments. We propose a mechanism by which mutant desmin slows desmin remodeling in myocytes by retaining nebulin near the Z-discs. On the basis of these data, we suggest that for some filament-forming desmin mutants, the molecular etiology of desminopathy results from subtle deficiencies in their association with nebulin, a major actin-binding filament protein of striated muscle.

Nebulin module 164 borders one side of the Z-disc in cardiomyocytes. (A) The layouts of desmin and nebulin. Desmin is composed of an α-helical coiled-coil rod central region bordered by non–α-helical head and tail regions. It is assumed that the rod of desmin is subdivided into a pre-coil domain (pcd) and coil 1A, linker L1, coil 1B, linker L12, and coil 2 domains, as reported for vimentin (). Shown is the localization of desminopathy desmin mutations, S46F/Y (head), E245D (coil 1B), and T453I (tail), and the random mutation K190A (coil 1B) used in this study. Nebulin contains 184 actin-binding single modules (M), short segments formed by 35–amino acid tandem repeat containing the SDxxYK motif. The modules are organized into superrepeats formed by seven modules containing the WLKGIGW motif. The N-terminal end of nebulin is rich in glutamic acid (acidic region), with a SH3 domain near its C-terminus. The nebulin modules that are important for interaction with desmin are shown in yellow (), and the ones that have the highest affinity for desmin are shown in red (). (B) Canine muscle tissues were costained with nebulin M164 (green) and desmin (red). Although desmin was detected in all three types of muscle analyzed (b, e, h), these images show that nebulin's intensity is the highest in skeletal muscle (a) and is expressed in low amounts in cardiac or smooth muscles (d, g). Bar, 10 μm.

Distribution patterns of desmin and nebulin in primary myocytes and muscle tissues. (A) Primary sequence analysis of the 22mer peptide within nebulin's module 164 (5766–5787) used to generate the custom peptide antibody. Blastp and ClustalW (version 1.83) alignments of this peptide show a highly conserved region in nebulin for the different species analyzed (human, apes, canines, and rodents). In contrast, this peptide only had 54% identical amino acids when aligned to chick nebulin. (B) Rat psoas skeletal muscle showed striking desmin (red) striations that appear to encircle each Z-disc, whereas nebulin M164 (green) is closely located on or along these desmin striations (see inset). Bar, 10 μm. (C) Primary cardiomyocytes costained for nebulin M164 (green) and Z-disc marker α-actinin (red) show intense nuclear staining. Moreover, the α-actinin staining shows clear striations at the Z-discs of the sarcomeres of muscle cells, whereas nebulin M164 was enriched at the periphery of the Z-discs. The inset shows filaments of nebulin projecting outward in both directions from the Z-discs. Bar, 10 μm. (D) Recombinant nebulin M160–164 is recognized by peptide antibody against nebulin module 164 and does not cross-react with nebulette M1–5. Immunoblots and corresponding total protein stain (Ponceau S, lanes 1, 3, and 5) of N-terminal His-tagged nebulin M160–164 recombinant protein (lanes 1–4) or nebulette M1–5 (lanes 5 and 6) were probed using standard Western blot conditions with the following antibodies: anti–nebulin M164 antibodies (lanes 2 and 6) or commercial anti-His antibodies (lane 4). Blots detect a ∼21-kDa band corresponding to the expected size of the recombinant nebulin M160–164 fragmentin strips containing nebulin, but little or no reactivity was found in the strip containing nebulette M1–5.

Desmin contains multiple binding sites for nebulin. High-speed cosedimentation was used to determine whether a specific desmin domain preferentially bound to a nebulin fragment. Four recombinant proteins comprising one (rod, headless, tailless) or all desmin domains were purified and coassembled with a nebulin recombinant fragment encompassing modules 160–164 for 1 h at 37°C under buffer conditions that facilitated desmin filament assembly. (A) Coomassie-stained SDS–PAGE gels show each sample fractionated into total (T), supernatant (Sup), sucrose (Suc), and pellet (P). Whereas headless, rod, and nebulin are largely soluble, both full-length and tailless desmin sedimented in the pellet (top). When nebulin M160–164 was added to full-length desmin or any of the desmin truncation proteins tested, all of them bound, as demonstrated by nebulin cosedimenting with each protein. (B) Schematic representation of desmin fragments used in this assay.

Nebulin M160–164 binds to desmin oligomers. The association between nebulin M160–164 and desmin oligomers was evaluated by sedimentation velocity analysis at 20°C in a 0.2 mM sodium phosphate (pH 7.5) buffer system. (A) At 40,000 rpm, a strong interaction between desmin and nebulin M160–164 was detected when a 10-fold excess of nebulin in relation to desmin (100 μM nebulin plus 9.3 μM desmin) was used. This is shown by ∼50% decrease for nebulin in the area under the curve for the mixed sample (red plot) as compared with nebulin alone (blue plot). Under these conditions desmin sediments at ∼11 S, as previously reported (black). (B) At 20,000 rpm, different molar ratios of nebulin in relation to desmin alone (black) were used as follows: 0.9 (blue), 2.3 (green), 4.7 (cyan), 9.3 (red), and 0 (pink) μM. A twofold excess of nebulin in relation to desmin significantly reduced the area under the desmin curve at ∼11 S (pink), as compared with a 1:1 ratio (red), suggesting a decrease in the concentration of free desmin oligomers. (C) At 30,000 rpm, for a molar ratio of desmin to nebulin of 1:2 (7.5 μM desmin to 16 μM nebulin), the desmin–nebulin complex exhibited a broad sedimentation velocity between ∼20 and 30 S, as evidenced from the dark blue curve imposed above the sedimentation profile for desmin alone (aqua). For comparison, we also evaluated nebulin alone (green).

Nebulin binding delays the assembly of mutant desmin into filaments. Cosedimentation assays evaluated the efficiency of WT and mutant desmin interaction with nebulin under conditions that promote desmin dimer and tetramer formation or filament assembly. (A) Right, a cosedimentation assay in proteins dialyzed in 5 mM Tris (pH 7.4) at a molar ratio of 1:1 without salt; left, a parallel assay under salt conditions that promote desmin filament assembly (+100 mM NaCl). In both cases, the proteins were incubated for 1 h at 37°C before ultracentrifugation. (B) The amount of desmin or nebulin recovered in pellets was quantified by band densitometry using ImageJ software. Plotted are the averaged and standard deviations obtained from three independent experiments. Bar graphs show the percentage of protein recovered in the pellets in the absence (black bars) or presence (white bars) of salt. Coassembled fractions are plotted in the third bar (intensity measured for desmin) and fourth bar (intensity measured for nebulin) for each condition.

Delayed kinetic profile of mutant desmin E245D associated with nebulin M160–164. Recombinant nebulin M160–164 binds strongly to WT and mutant desmin E245D filaments assembled in vitro. (A) Kinetic cosedimentation assays were performed using assembly conditions that promoted desmin filament formation. (B) Gel band densities in the pellet were measured for desmin (a) and nebulin bands (b) using ImageJ and plotted as a function of time. The pelleting behavior of nebulin when coincubated with mutant desmin E245D was significantly delayed as compared with WT desmin (compare red to black plots in , b). Values plotted show the average of three independent experiments.

Mutant desmin binds to nebulin M160–164 with higher affinity and capacity in a pH-dependent manner. The binding of a nebulin M160–164 to WT desmin or mutant desmin (S46Y, E245D, and T453I) was compared in ELISAs at pH 7.4 and 8.4. Graphs show the normalized averaged binding to nebulin for WT and each mutant under the same experimental conditions. Curves show the nonlinear fitted averages of seven or eight experiments for WT desmin and two or three experiments for mutant desmin using the Michaelis–Menten equation. Values show the means ± SD. (A) ELISAs show increased binding capacities and affinities for all mutants tested as compared with WT when desmin proteins were dialyzed to 5 mM Tris-HCl, 1 mM EDTA, 1 mM EGTA, pH 7.4. (B) No difference in binding capacities and affinities for nebulin were found for mutants as compared with WT when the indicated desmin proteins were dialyzed to 5 mM Tris-HCl, 1 mM EDTA, 1 mM EGTA, pH 8.4. lists the averages of all the measured Kd and Bmax values obtained for each experiment.

Dynamics of desmin and thin filament proteins in live cardiomyocytes. FRAP compares the dynamics of GFP-nebulin M163-170 to GFP-tropomodulin (Tmod), and desmin GFP-coil 1B to its mutants K190A and E245D in live cardiomyoctes. (A) The live image of a cardiomyocyte shows the recovery of GFP-Tmod fluorescence after photobleaching. The arrows point to the photobleached region of interest on a myofibril, at the pointed ends of the thin filaments, near the M-lines of the sarcomeres. (B, D) Fluorescence intensities were normalized to bleach level of zero, and the recoveries of fluorescence were plotted as a function of time after bleaching. The best-fit values of the fluorescence recoveries were a one-phase association equation from which half-lives (t1/2) were calculated. (C, E) Bar graphs comparing the averaged half-lives (t1/2) obtained for each protein. The calculated mean recovery half-life is as follows: for GFP, 0.9 ms; for the nebulin fragment, 2.3 ms; for Tmod, 1.3 ms; for coil 1B, 1.2 ms; for coil 1B K190A, 1.1 ms; and for coil 1B E245D, 1.7 ms. The data show that nebulin, tropomodulin, and mutant desmin GFP-coil 1B E245D are significantly slower relative to cells expressing GFP alone, whereas insignificant differences were measured for GFP-coil 1B or its K190A mutant. The number of cells analyzed per sample is indicated (n) under each column. Values show the means ± SD. Asterisks indicate significant differences relative to cells expressing GFP alone (*p < 0.05; **p < 0.01; Student's unpaired t test).

A model for the association of nebulin binding to mutant desmin. In this model, mutant desmins competent for filament assembly bind strongly to nebulin with altered stoichiometry, slowing the dynamics of desmin turnover, retaining and retarding nebulin mobility at the ends of the actin thin filament near the Z-bands of the sarcomeres (DIFs are depicted as cylinders). We predict that nebulin changes its conformation upon binding to mutant desmin (note that nebulin is represented as yellow circles when bound to WT desmin and as hexagons when bound to mutant desmin). We propose that the altered association indirectly and ultimately leads to a progressive loss of actin filament stability, interfering with actin–myosin cross-bridge kinetics. For clarity, the WT desmin (blue) and mutant desmin rods (red) and the nebulin M160–164 modules (rather than the multimodular full-length molecule) are drawn.